Name: lipid supplementation for longevity and gene transcriptional analysis in caenorhabditis elegans
Uploaded: 2022-12-09T13:05:00
Description: Watch this Scientific Journal Video about Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans at JoVE.com

我们感谢 P. Svay 的维护支持。这项工作得到了NIH资助R01AG045183（MCW），R01AT009050（MCW），R01AG062257（MCW），DP1DK113644（MCW），March of Dimes基金会（MCW），韦尔奇基金会（MCW），HHMI研究员（MCW）和NIH T32 ES027801博士前学生研究员（MS）的支持。一些菌株由CGC提供，CGC由NIH研究基础设施计划办公室（P40 OD010440）资助。

图1 描述了使用不同实验设置的脂质喂养示意图。
1. 脂质条件细菌的制备
<ol><li>通过将 5.85 g NaCl、1.0 g K 2 HPO 4 和 6.0 g KH2PO4（参见材料表）溶解在 999 mL 去离子水中来制备细菌稀释饮食限制 （BDR）碱溶液。用0.5M KOH将pH调节至6.0，然后通过0.22μm过滤器过滤。 注意：BDR溶液可以在室温下储存，?...

<ol>
	<li>Fahy, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+of+the+LIPID+MAPS+comprehensive+classification+system+for+lipids+1.">Update of the LIPID MAPS comprehensive classification system for lipids 1.</a> Journal of Lipid Research. 50, 9-14 (2009).</li><li>Liebisch, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+LIPID+MAPS+classification,+nomenclature,+and+shorthand+notation+for+MS-derived+lipid+structures.">Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures.</a> Journal of Lipid Research. 61 (12), 1539-1555 (2020).</li><li>Mutlu, A. S., Duffy, J., Wang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+and+lipid+signals+in+aging+and+longevity.">Lipid metabolism and lipid signals in aging and longevity.</a> Developmental Cell. 56 (10), 1394-1407 (2021).</li><li>Kimura, T., Jennings, W., Epand, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+specific+lipid+species+in+the+cell+and+their+molecular+mechanism.">Roles of specific lipid species in the cell and their molecular mechanism.</a> Progress in Lipid Research. 62, 75-92 (2016).</li><li>Duffy, J., Mutlu, A. S., Wang, M. C., Olsen, A., Gill, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Metabolism,+Lipid+Signalling+and+Longevity.">Lipid Metabolism, Lipid Signalling and Longevity.</a> Ageing: Lessons from C. elegans. Healthy Ageing and Longevity. , 307-329 (2017).</li><li>Lesa, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+chain+poly-unsaturated+fatty+acids+are+required+for+efficient+neurotransmission+in+C.+elegans.">Long chain poly-unsaturated fatty acids are required for efficient neurotransmission in C. elegans.</a> Journal of Cell Science. 116 (24), 4965-4975 (2003).</li><li>Folick, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+signaling+molecules+regulate+longevity+in+Caenorhabditis+elegans.">Lysosomal signaling molecules regulate longevity in Caenorhabditis elegans.</a> Science. 347 (6217), 83-86 (2015).</li><li>Ramachandran, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+signaling+promotes+longevity+by+adjusting+mitochondrial+activity.">Lysosomal signaling promotes longevity by adjusting mitochondrial activity.</a> Developmental Cell. 48 (5), 685-696 (2019).</li><li>Byrne, E. F. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+Smoothened+regulation+by+its+extracellular+domains.">Structural basis of Smoothened regulation by its extracellular domains.</a> Nature. 535 (7613), 517-522 (2016).</li><li>Corsi, A. K., Wightman, B., Chalfie, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+window+into+biology:+a+primer+on+Caenorhabditis+elegans.">Transparent window into biology: a primer on Caenorhabditis elegans.</a> Genetics. 200 (2), 387-407 (2015).</li><li>Nigon, V. M., F&#233;lix, M. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+research+on+C.+elegans+and+other+free-living+nematodes+as+model+organisms.">History of research on C. elegans and other free-living nematodes as model organisms.</a> WormBook. , 1-84 (2017).</li><li>Kutscher, L. M., Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+mutagenesis+in+C.+elegans.">Forward and reverse mutagenesis in C. elegans.</a> WormBook. , 1-26 (2014).</li><li>Brooks, K. K., Liang, B., Watts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+bacterial+diet+on+fat+storage+in+C.+elegans.">The influence of bacterial diet on fat storage in C. elegans.</a> PloS ONE. 4 (10), 7545 (2009).</li><li>Brock, T. J., Browse, J., Watts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+desaturation+and+the+regulation+of+adiposity+in+Caenorhabditis+elegans.">Fatty acid desaturation and the regulation of adiposity in Caenorhabditis elegans.</a> Genetics. 176 (2), 865-875 (2007).</li><li>Brock, T. J., Browse, J., Watts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+unsaturated+fatty+acid+composition+in+C.+elegans.">Genetic regulation of unsaturated fatty acid composition in C. elegans.</a> PloS Genetics. 2 (7), 108 (2006).</li><li>Watts, J. L., Phillips, E., Griffing, K. R., Browse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiencies+in+C20+poly-unsaturated+fatty+acids+cause+behavioral+and+developmental+defects+in+Caenorhabditis+elegans+fat-3+mutants.">Deficiencies in C20 poly-unsaturated fatty acids cause behavioral and developmental defects in Caenorhabditis elegans fat-3 mutants.</a> Genetics. 163 (2), 581-589 (2003).</li><li>Watts, J. L., Browse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+poly-unsaturated+fatty+acid+synthesis+in+Caenorhabditis+elegans.">Genetic dissection of poly-unsaturated fatty acid synthesis in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences. 99 (9), 5854-5859 (2002).</li><li>Watts, J. L., Browse, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoyl-CoA-specific+&#916;9+fatty+acid+desaturase+from+Caenorhabditis+elegans.">Palmitoyl-CoA-specific &#916;9 fatty acid desaturase from Caenorhabditis elegans.</a> Biochemical and Biophysical Research Communications. 272 (1), 263-269 (2000).</li><li>Watts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+synthesis+and+adiposity+regulation+in+Caenorhabditis+elegans.">Fat synthesis and adiposity regulation in Caenorhabditis elegans.</a> Trends in Endocrinology &amp; Metabolism. 20 (2), 58-65 (2009).</li><li>Peyou-Ndi, M. M., Watts, J. L., Browse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+an+animal+&#916;12+fatty+acid+desaturase+gene+by+heterologous+expression+in+Saccharomyces+cerevisiae.">Identification and characterization of an animal &#916;12 fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae.</a> Archives of Biochemistry and Biophysics. 376 (2), 399-408 (2000).</li><li>Spychalla, J. P., Kinney, A. J., Browse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+animal+&#969;-3+fatty+acid+desaturase+by+heterologous+expression+in+Arabidopsis.">Identification of an animal &#969;-3 fatty acid desaturase by heterologous expression in Arabidopsis.</a> Proceedings of the National Academy of Sciences. 94 (4), 1142-1147 (1997).</li><li>Watts, J. L., Browse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+a+&#916;5-fatty+acid+desaturase+from+Caenorhabditis+elegans.">Isolation and characterization of a &#916;5-fatty acid desaturase from Caenorhabditis elegans.</a> Archives of Biochemistry and Biophysics. 362 (1), 175-182 (1999).</li><li>Deline, M. L., Vrablik, T. L., Watts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+supplementation+of+polyunsaturated+fatty+acids+in+Caenorhabditis+elegans.">Dietary supplementation of polyunsaturated fatty acids in Caenorhabditis elegans.</a> Journal of Visualized Experiments. (81), e50879 (2013).</li><li>Estes, R. E., Lin, B., Khera, A., Davis, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+influence+on+neurodegenerative+disease+progression:+is+the+vehicle+as+important+as+the+cargo.">Lipid metabolism influence on neurodegenerative disease progression: is the vehicle as important as the cargo.</a> Frontiers in Molecular Neuroscience. 14, 788695 (2021).</li><li>Sunshine, H., Iruela-Arispe, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+lipids+and+cell+signaling.">Membrane lipids and cell signaling.</a> Current Opinion in Lipidology. 28 (5), 408-413 (2017).</li><li>Han, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mono-unsaturated+fatty+acids+link+H3K4me3+modifiers+to+C.+elegans+lifespan.">Mono-unsaturated fatty acids link H3K4me3 modifiers to C. elegans lifespan.</a> Nature. 544 (7649), 185-190 (2017).</li><li>O'Rourke, E. J., Kuballa, P., Xavier, R., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#969;-6+Poly-unsaturated+fatty+acids+extend+life+span+through+the+activation+of+autophagy.">&#969;-6 Poly-unsaturated fatty acids extend life span through the activation of autophagy.</a> Genes &amp; Development. 27 (4), 429-440 (2013).</li><li>Steinbaugh, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-mediated+regulation+of+SKN-1/Nrf+in+response+to+germ+cell+absence.">Lipid-mediated regulation of SKN-1/Nrf in response to germ cell absence.</a> eLife. 4, 07836 (2015).</li><li>Blackwell, T. K., Steinbaugh, M. J., Hourihan, J. M., Ewald, C. Y., Isik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKN-1/Nrf,+stress+responses,+and+aging+in+Caenorhabditis+elegans.">SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans.</a> Free Radical Biology and Medicine. 88, 290-301 (2015).</li><li>Ezzili, C., Otrubova, K., Boger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+amide+signaling+molecules.">Fatty acid amide signaling molecules.</a> Bioorganic &amp; Medicinal Chemistry Letters. 20 (20), 5959-5968 (2010).</li><li>Qi, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#969;-3+fatty+acid+&#945;-linolenic+acid+extends+Caenorhabditis+elegans+lifespan+via+NHR-49/PPAR&#945;+and+oxidation+to+oxylipins.">The &#969;-3 fatty acid &#945;-linolenic acid extends Caenorhabditis elegans lifespan via NHR-49/PPAR&#945; and oxidation to oxylipins.</a> Aging Cell. 16 (5), 1125-1135 (2017).</li><li>Shemesh, N., Meshnik, L., Shpigel, N., Ben-Zvi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary-induced+signals+that+activate+the+gonadal+longevity+pathway+during+development+regulate+a+proteostasis+switch+in+Caenorhabditis+elegans+adulthood.">Dietary-induced signals that activate the gonadal longevity pathway during development regulate a proteostasis switch in Caenorhabditis elegans adulthood.</a> Frontiers in Molecular Neuroscience. 10, 254 (2017).</li><li>Savini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosome+lipid+signalling+from+the+periphery+to+neurons+regulates+longevity.">Lysosome lipid signalling from the periphery to neurons regulates longevity.</a> Nature Cell Biology. 24 (6), 906-916 (2022).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , (2006).</li><li>Wang, M. C., O'Rourke, E. J., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+metabolism+links+germline+stem+cells+and+longevity+in+C.+elegans.">Fat metabolism links germline stem cells and longevity in C. elegans.</a> Science. 322 (5903), 957-960 (2008).</li><li>Jacob, T. C., Kaplan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EGL-21+carboxypeptidase+E+facilitates+acetylcholine+release+at+Caenorhabditis+elegans+neuromuscular+junctions.">The EGL-21 carboxypeptidase E facilitates acetylcholine release at Caenorhabditis elegans neuromuscular junctions.</a> The Journal of Neuroscience. 23 (6), 2122-2130 (2003).</li><li>Kass, J., Jacob, T. C., Kim, P., Kaplan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EGL-3+proprotein+convertase+regulates+mechanosensory+responses+of+Caenorhabditis+elegans.">The EGL-3 proprotein convertase regulates mechanosensory responses of Caenorhabditis elegans.</a> The Journal of Neuroscience. 21 (23), 9265-9272 (2001).</li><li>Bael, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+evidence+for+neuropeptide-amidating+enzymes+in+Caenorhabditis+elegans.">Mass spectrometric evidence for neuropeptide-amidating enzymes in Caenorhabditis elegans.</a> Journal of Biological Chemistry. 293 (16), 6052-6063 (2018).</li><li>Fu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+metabolic+fingerprinting+of+neutral+lipids+with+hyperspectral+stimulated+Raman+scattering+microscopy.">In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy.</a> Journal of the American Chemical Society. 136 (24), 8820-8828 (2014).</li></ol>

脂质补充剂已被用于衰老研究，以阐明某些脂质物种对健康衰老的直接影响6，7，23，26，27，31。然而，脂质补充过程可能具有挑战性，实验之间的任何不一致都可能导致不可重复的结果。在这里，记录了第一个详细的分步协议，以指导新科学家避?...

血脂 脂质是可溶于有机溶剂但不溶于水的小疏水性或两亲性分子1，2。不同的脂质分子根据其链中所含的碳数量、位置、双键数量和结合结构（包括甘油或磷酸盐）相互区分。脂质在不同细胞内和跨不同细胞发挥关键作用，以调节生物体功能，包括构成膜双层、提供能量储存和充当信号分子3，4。
首先，脂质是生物膜的结构成分，包括将内部隔室与细胞外环境分开的质膜和细胞内亚细胞膜。其次，脂质是脊椎动物和无脊椎动物能量储存的主要形式。中性脂质，包括三酰基甘油，在各种组织中长时间储存，包括脂肪组织。在线虫秀丽隐杆线虫中，肠道是主要的代谢脂肪储存器官;它的功能不仅参与营养物质的消化和吸收，还参与解毒过程，类似于哺乳动物肝细胞的活性。其他脂肪储存组织包括种系，其中脂质对卵母细胞发育至关重要，以及由皮肤样表皮细胞组成的皮下组织3，5。第三，近年来，越来越多的证据表明，脂质是参与细胞内和细胞外信号传导的强大信号分子，通过直接作用于多种受体，包括G蛋白偶联和核受体，或通过膜流动性调节或翻译后修饰间接作用6，7，8，9.进一步的研究将继续阐明脂质信号传导在促进长寿和健康方面的潜在分子机制。
模式生物对于解决在人类中研究的复杂特定生物学问题非常重要。例如，秀丽隐杆线虫是进行遗传分析以剖析与人类营养和疾病相关的生物过程的绝佳模型10。与人体生理学、复杂组织、行为模式和丰富的遗传操作工具相关的高度保守的分子途径使秀丽隐杆线虫成为非凡的模式生物11.例如，秀丽隐杆线虫在转发遗传筛选以识别表型特异性基因方面非常出色，以及通过RNA干扰12进行全基因组反向遗传筛选。
在实验室中，线虫生长在琼脂培养皿上，琼脂培养皿上接种了大肠杆菌草坪，提供蛋白质、碳水化合物、饱和和不饱和脂肪酸等常量营养素作为能量和构建块的来源，以及辅因子和维生素等微量营养素13。与哺乳动物类似，线虫从棕榈酸和硬脂酸（分别为饱和的16碳和18碳分子）合成脂肪酸分子，这些分子依次去饱和并拉长为多种单不饱和脂肪酸（MUFA）和多不饱和脂肪酸（PUFA）14，15，16，17，18。有趣的是，秀丽隐杆线虫能够从头合成所有必需的脂肪酸和参与脂肪酸生物合成、去饱和和伸长的核心酶，促进长链多不饱和脂肪酸的合成19。与其他动物物种不同，秀丽隐杆线虫可以用自己的ω-3去饱和酶将18碳和20碳ω-6脂肪酸转化为ω-3脂肪酸。此外，蠕虫具有Δ12去饱和酶，可催化油酸（OA，18：1）形成亚油酸（LA）20，21。大多数动物或植物同时缺乏Δ12和ω-3去饱和酶，因此依靠膳食摄入ω-6和ω-3来获得多不饱和脂肪酸，而秀丽隐杆线虫不需要膳食脂肪酸22。缺乏功能性去饱和酶的分离突变体已被用于研究特定脂肪酸在不同生物过程中的功能，包括繁殖、生长、寿命和神经传递。单个脂肪酸对特定生物学途径的影响可以使用遗传方法和饮食补充剂来解决16，17，23。迄今为止，脂质研究的重点是表征参与神经和发育条件下脂质合成、降解、储存和分解的基因24.然而，脂质在长寿调节中的作用才刚刚开始显现。
长寿调节中的脂质信号传导 脂质通过激活不同组织和细胞类型中的细胞信号级联，在长寿调节中起着至关重要的作用。最近的研究强调了脂质在通过脂质结合蛋白或识别膜受体 调节 转录和细胞间通讯中的积极作用25。此外，膳食脂质补充剂为剖析脂质代谢如何影响 秀丽隐杆线虫的寿命提供了极好的工具。不同的MUFAs和PUFAs已被证明通过激活转录因子来促进长寿26，27。
长寿模型，包括胰岛素/IGF-1信号传导和种系前体细胞的消融，与MUFA生物合成途径有关，MUFA补充剂，包括油酸，棕榈油酸和顺式牛痘，足以延长秀丽隐杆线虫的寿命26。尽管MUFA管理赋予的长寿效应需要进一步研究，但其潜在机制很可能由SKN-1 / Nrf2转录因子介导，SKN-1 / Nrf2转录因子是氧化应激反应和长寿调节的关键激活剂28，29。在MUFAs中，一类称为N-酰基乙醇胺（NAEs）的特定类别的脂肪酰基乙醇酰胺在不同的机制中起着至关重要的作用，包括炎症，过敏，学习，记忆和能量代谢30。特别是，称为油酰乙醇酰胺（OEA）的脂质分子已被确定为长寿的正调节因子，通过促进脂质结合蛋白8（LBP-8）易位到细胞核中以激活核激素受体NHR-49和NHR-807。补充OEA类似物KDS-5104足以延长寿命，并诱导参与氧化应激反应和线粒体β氧化的基因表达7，8。
同时，PUFA的作用也与长寿调节有关。给予PUFA ω-3脂肪酸α-亚麻酸（ALA）通过激活NHR-49 / PPARα，SKN-1 / NRF转录因子和诱导线粒体β氧化来促进长寿31。有趣的是，ALA的过氧化物产物，称为脂氧化物，激活SKN-1 / NRF，表明PUFAs及其氧化衍生物都可以带来长寿益处23。补充ω-6脂肪酸花生四烯酸（AA）和二同γ-亚麻酸（DGLA）通过自噬活化 延长 寿命，促进蛋白质质量控制，并导致浪费和有毒蛋白质聚集体的降解27，32。最近，由脂质结合蛋白3（LBP-3）和DGLA介导的细胞非自主信号传导调节已被证明通过向神经元发送外周信号来促进长寿至关重要，这表明脂质分子在系统水平的组织间通讯中具有长期作用33。本研究报告了用接种在平板上的细菌或液体培养中的细菌悬浮液进行脂质补充的每个步骤。这些方法用于评估寿命和转录分析，使用全身内容物或来自少数蠕虫的解剖组织。以下技术可以适用于各种营养研究，并提供有效的工具来剖析脂质代谢如何影响长寿和健康老龄化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Pestle</td>
			<td>Genesee Scientific</td>
			<td>93-165P15</td>
			<td>For worm grinding with Trizol</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9639-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>AmfiRivert cDNA Synthesis Platinum Master Mix</td>
			<td>GenDEPOT</td>
			<td>R5600</td>
			<td>For reverse transcription from bulk worm samples</td>
		</tr>
		<tr>
			<td>Applied Biosystems QuanStudio 3 Real-Time PCR</td>
			<td>ThermoFisher</td>
			<td>A28567</td>
			<td>For qRT-PCR</td>
		</tr>
		<tr>
			<td>Benchmark Scientific StripSpin 12 Microcentrifuge</td>
			<td>Benchmark Scientific</td>
			<td>C1248</td>
			<td>For spin down PCR tubes</td>
		</tr>
		<tr>
			<td>Branson 450 Digital Sonifier, w/ 1/8&#34; tip</td>
			<td>Branson Ultrasonic Corporation</td>
			<td>100-132-888R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8503-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424 R centrifuge</td>
			<td>Eppendorf</td>
			<td>22620444R</td>
			<td>For RNA extraction</td>
		</tr>
		<tr>
			<td>Eppendorf vapo protect mastercycler pro</td>
			<td>Eppendorf</td>
			<td>950030010</td>
			<td>For reverse transcription</td>
		</tr>
		<tr>
			<td>Ethanol, Absolute (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Greiner Bio-One CELLSTAR, 12 W Plate</td>
			<td>Neta Scientific</td>
			<td>665180</td>
			<td>12-well plates for licuid feeding</td>
		</tr>
		<tr>
			<td>Greiner Bio-One Petri Dish, Ps, 100 x 20 mm</td>
			<td>Neta Scientific</td>
			<td>664161</td>
			<td>For bacterial LB plates and worm 10-cm NGM plates</td>
		</tr>
		<tr>
			<td>Greiner Bio-One Petri Dish, Ps, 60 x 15 mm</td>
			<td>Neta Scientific</td>
			<td>628161</td>
			<td>For worm6-cm NGM plates</td>
		</tr>
		<tr>
			<td>Invitrogen nuclease-free water</td>
			<td>ThermoFisher</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproanol</td>
			<td>Sigma</td>
			<td>PX1835-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole hydrochloride</td>
			<td>VWR</td>
			<td>SPCML1054</td>
			<td></td>
		</tr>
		<tr>
			<td>lipl-4Tg</td>
			<td>MCW Lab</td>
			<td>N/A</td>
			<td>Transgenic C. elegans</td>
		</tr>
		<tr>
			<td>lipl-4Tg;fat-3(wa22)</td>
			<td>MCW Lab</td>
			<td>N/A</td>
			<td>Transgenic C. elegans</td>
		</tr>
		<tr>
			<td>Luria Broth Base</td>
			<td>ThermoFisher</td>
			<td>12795-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma</td>
			<td>M2643-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp EnduraPlate Optical 96-Well Fast Clear Reaction Plate with Barcode</td>
			<td>ThermoFisher</td>
			<td>4483354</td>
			<td>96-well qPCR plate</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Applied BioSystem</td>
			<td>4311971</td>
			<td>For sealing the 96-well qPCR plate</td>
		</tr>
		<tr>
			<td>Milli-Q Advantage A10 Water Purification System</td>
			<td>Sigma</td>
			<td>Z00Q0V0WW</td>
			<td>Deionized water used to make all reagents, including buffer and cultural media, unless specified as nuclease-free water in the protocol</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N/A</td>
			<td>C. elegans wild isolate</td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000 Spectrophotometer</td>
			<td>ThermoFisher</td>
			<td>N/A</td>
			<td>For measuring RNA concentration</td>
		</tr>
		<tr>
			<td>OP50</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N/A</td>
			<td>Bacteria used as C. elegans food</td>
		</tr>
		<tr>
			<td>Potasium phosphate dibasic trihydrate (K2HPO4&#183;3H2O)</td>
			<td>Sigma</td>
			<td>P5504-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium phosphate monobasic (KH2PO4)</td>
			<td>Sigma</td>
			<td>P0662-2.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green cells-to-Ct kit</td>
			<td>ThermoFisher</td>
			<td>4402953</td>
			<td>For reverse transcription and qPCR from a few worms or worm tissue</td>
		</tr>
		<tr>
			<td>Power SYBR Green Master Mix</td>
			<td>ThermoFisher</td>
			<td>4367659</td>
			<td>For qPCR from bulk worm samples</td>
		</tr>
		<tr>
			<td>Pure Bright germicidal ultra bleach&#160;</td>
			<td>KIK International LLC.</td>
			<td>59647210143</td>
			<td>6% house bleach For worm egg preparation</td>
		</tr>
		<tr>
			<td>Pyrex spot plate with nine depressions</td>
			<td>Sigma</td>
			<td>CLS722085-18EA</td>
			<td>Watch glass for dissecting the worms</td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>ThermoFisher</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cloride (NaCl)</td>
			<td>Sigma</td>
			<td>S7653-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma</td>
			<td>SX0590-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic heptahydrate (Na2HPO4&#183;7H2O)</td>
			<td>Sigma</td>
			<td>S9390-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Sorvall Legend Mach 1.6R Centrifuge</td>
			<td>Thermo</td>
			<td>7500-4337</td>
			<td>For bacteria collection</td>
		</tr>
		<tr>
			<td>Thermo Sorvall ST 8 centrifuge</td>
			<td>Thermo</td>
			<td>7500-7200</td>
			<td>For worm egg preparation</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>TheroFisher</td>
			<td>15596018</td>
			<td>RNA extraction reagent</td>
		</tr>
		<tr>
			<td>Turbo DNA-free kit</td>
			<td>ThermoFisher</td>
			<td>AM1907</td>
			<td>For removing DNA contamination in RNA extractions</td>
		</tr>
		<tr>
			<td>Vortexer 59</td>
			<td>Denville Scientific INV</td>
			<td>S7030</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Disposable Pellet Mixers and Cordless Motor</td>
			<td>VWR</td>
			<td>47747-370</td>
			<td>For worm grinding with Trizol</td>
		</tr>
		<tr>
			<td>VWR Kinetic Energy 26 Joules Mini Centrifuge C1413 V-115</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>For worm collection. Discontinued model, a similar one available at VWR with Cat# 76269-064</td>
		</tr>
		<tr>
			<td>Worm picker</td>
			<td>WormStuff</td>
			<td>59-AWP</td>
			<td></td>
		</tr>
	</tbody>
</table>

lipid supplementation for longevity and gene transcriptional analysis in caenorhabditis elegans

衰老是一个复杂的过程，其特征是由环境和遗传贡献引起的渐进式生理变化。脂质在构成细胞膜的结构成分、储存能量和作为信号分子方面至关重要。脂质代谢和信号传导的调节对于激活不同的长寿途径至关重要。 秀丽隐杆线虫 是一种优秀而强大的生物体，可以剖析脂质代谢和信号传导对长寿调节的贡献。多项研究描述了特定脂质分子的饮食补充如何延长 秀丽隐杆线虫 的寿命;然而，补充条件的微小差异可能会导致不同实验室的科学家之间的可重复性问题。在这里，报道了秀 丽隐杆线虫 的两种详细补充方法，采用脂质补充，将细菌接种在平板上或在液体培养物中细菌悬浮液。本文还提供了使用全蠕虫裂解物或来自少数蠕虫的解剖组织进行终身脂质补充和qRT-PCR分析的寿命测定的详细信息。结合纵向研究和脂质补充转录研究，喂养测定提供了可靠的方法来剖析脂质如何影响长寿和健康老龄化。该方法还可以适用于各种营养筛查方法，以使用少量解剖组织或少数动物来评估转录本子集的变化。

在补充脂质时使用一些全虫验证转录变化 为了研究从几个全蠕虫中提取和逆转录RNA到cDNA的方案是否可重现并与来自散装蠕虫的数据进行比较，采用了一种在肠道中过表达溶酶体酸性脂肪酶lipl-4的长寿命蠕虫菌株7，8，33，35。先前研究7，8</...

Watch this Scientific Journal Video about Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans at JoVE.com

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

本协议描述了 秀丽隐杆线虫液体和平板培养物中的脂质补充方法，以及来自大量或少数蠕虫和蠕虫组织的纵向研究和基因转录分析。

秀丽隐杆线虫长寿的脂质补充和基因转录分析

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are ...

该协议描述了两种脂质补充策略，结合了模式生物秀丽隐杆线虫的纵向和转录分析。该技术描述了如何使用少量蠕虫或解剖的蠕虫组织检查转录变化，以及如何将昆虫种群的液体喂养与水力技术相结合。执行此技术的个人必须练习组织解剖，因为执行实验的时间窗口很短，以实现足够的转录分析。首先，通过在室温下以4， 000G离心过夜生长的培养物10分钟来收集OP50细菌。弃去上清液。通过涡旋将细菌沉淀悬浮在20毫升细菌稀释液，饮食限制或BDR碱中，再次重复离心10分钟。弃去上清液后，将细菌沉淀重新悬浮在BDR培养基中以制备20X BDR细菌储备液。使用前使用BDR培养基将细菌原液稀释至5X。以乙醇或二甲基亚砜为溶剂，在新的高压灭菌玻璃小瓶中制备脂质储备溶液，并在玻璃小瓶中填充氩气或氮气以防止氧化。将脂质储备溶液转移到BDR细菌溶液中，以在喂养条件下达到所需的最终浓度。通过涡旋20秒将其彻底混合。通过将相同体积的过滤乙醇或二甲基亚砜与BDR细菌混合来制备控制的载体。通过将所需量的脂质或载体对照转移到 12 孔板的每个孔中进行脂质补充和液体培养，每个喂养条件重复三到四个重复。将同步的秀丽隐杆线虫悬浮液与 5X BDR 细菌以一比一的比例混合，以达到每毫升 1， 500 条蠕虫和 2.5X 细菌的最终浓度。然后将两毫升蠕虫细菌混合物转移到12孔板的每个孔中。完成后，用箔纸包裹12孔板，并在20摄氏度的培养箱中以100 RPMM摇动板以获得所需的孵育长度。为了在平板上补充脂质，将一毫升脂质条件细菌接种到10厘米线虫生长培养基或NGM琼脂平板的中心。在使用大量板时，确保最终工作溶液在播种两个板之间多次涡旋。在生物安全罩内的黑暗中干燥板。将3000条蠕虫从同步蠕虫培养物转移到10厘米的平板上。在生物安全罩中干燥板，直到在补充脂质的平板上游泳的蠕虫开始爬行。干燥后，将脂质调节板在20摄氏度的培养箱中孵育所需的时间长度，如果使用多不饱和脂质，则保护板免受光照。通过在PCR管中将0.2微升DNase I与19.8微升裂解液混合，为每个样品制备20微升最终裂解溶液，并通过上下移液五次充分混合。为了从少量的全蠕虫中提取RNA，通过将15至20条蠕虫从细菌草坪转移到新鲜未播种的NGM板上来去除细菌。将15至20个蠕虫从未接种的NGM板转移到含有20微升新鲜制备的具有最少细菌数量的最终裂解溶液的PCR管中，并在室温下孵育裂解反应5分钟。孵育结束后，将样品保存在一个漂亮的冷水浴中，并探测蠕虫四次，每次五秒，每次振幅为30%，每次超声处理之间脉冲15秒。将试管在室温下孵育五分钟，然后将两微升终止溶液加入裂解反应中，并通过轻轻敲击混合。将反应在室温下孵育两分钟，然后将试管放在冰上。为了从蠕虫组织中提取RNA，从细菌草坪上挑选大约20条蠕虫，并将它们放入新鲜的未播种NGM板中以去除蠕虫中的细菌。将细菌尽可能少的20条蠕虫转移到含有500微升M9溶液的手表玻璃杯中，该表杯含有4微摩尔左旋咪唑。当蠕虫被固定时，使用可以连接到一毫升注射器的 25 号针头解剖种系或肠道。使用高压灭菌的玻璃移液管，将解剖的组织转移到PCR管中，并将管在冰上放置两分钟，使材料沉积在底部。在加入20微升终裂解溶液之前，从PCR管中取出上清液，并通过敲击将其充分混合。将裂解反应孵育五分钟后，加入两微升终止液并多次敲击试管。将试管孵育两分钟，然后再进行逆转录。在验证研究中，从大量种群和少数蠕虫中提取的RNA显示出神经肽加工基因egl-3和egl-21的相似诱导。这表明从少数蠕虫中提取RNA可以作为来自大量种群的标准CDNA合成技术的有效替代方案。在补充二高γ-亚麻酸（DGLA）的lipl-4转基因蠕虫的解剖肠中，未诱导神经元神经肽加工基因。不同浓度的DGLA补充剂挽救了Fat-3敲低后蠕虫寿命延长。补充脂质的蠕虫也可以用于RNA测序，蛋白质组学，代谢组学和行为研究，以识别这些特定条件下的其他表型。这种方法可以应用于衰老研究，脂质生物学和任何其他旨在建立某种表型与营养因子或代谢物之间关系的研究领域。

Research

JoVE Journal

Biology

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

科研

生物学

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

多不饱和脂肪酸的膳食补充剂<em&gt;秀丽隐杆线虫</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

线虫的核苷酸和蛋白质提取联合

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

卡诺尔哈布炎细胞外囊泡的纯化与分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...